Protein C is a member of the class of vitamin K-dependent serine protease coagulation factors. Protein C was originally identified for its anticoagulant and profibrinolytic activities. Protein C circulating in the blood is an inactive zymogen that requires proteolytic activation to regulate blood coagulation through a complex natural feedback mechanism. Human protein C is primarily made in the liver as a single polypeptide of 461 amino acids. This precursor molecule is then post-translationally modified by (i) cleavage of a 42 amino acid signal sequence, (ii) proteolytic removal from the one-chain zymogen of the lysine residue at position 155 and the arginine residue at position 156 to produce the two-chain form (i.e., light chain of 155 amino acid residues attached by disulfide linkage to the serine protease-containing heavy chain of 262 amino acid residues), (iii) carboxylation of the glutamic acid residues clustered in the first 42 amino acids of the light chain resulting in nine gamma-carboxyglutamic acid (Gla) residues, and (iv) glycosylation at four sites (one in the light chain and three in the heavy chain). The heavy chain contains the serine protease triad of Asp257, His211 and Ser360.
Similar to most other zymogens of extracellular proteases and the coagulation factors, protein C has a core structure of the chymotrypsin family, having insertions and an N-terminus extension that enable regulation of the zymogen and the enzyme. Of interest are two domains with amino acid sequences similar to epidermal growth factor (EGF). At least a portion of the nucleotide and amino acid sequences for protein C from human, monkey, mouse, rat, hamster, rabbit, dog, cat, goat, pig, horse, and cow are known, as well as mutations and polymorphisms of human protein C (see GenBank accession P04070). Other variants of human protein C are known which affect different biological activities.
Activation of protein C is mediated by thromblin, acting at the site between the arginine residue at position number 15 of the heavy chain and the leucine residue at position 16 (chymotrypsin numbering) (See Kisiel, J. Clin. Invest., 64:761-769, 1976; Marlar et al., Blood, 59:1067-1072, 1982; Fisher et al. Protein Science, 3:588-599, 1994). Other proteins including Factor Xa (Haley et al., J. Biol. Chem., 264:16303-16310, 1989), Russell's viper venom, and trypsin (Esmon et al., J. Biol. Chem., 251:2770-2776, 1976) also have been shown to enzymatically cleave and convert inactive protein C to its activated form.
Thrombin binds to thrombomodulin, a membrane-bound thrombin receptor on the luminal surface of endothelial cells, thereby blocking the procoagulant activity of thrombin via its exosite I, and enhancing its anticoagulant properties, i.e., activating protein C. As an anticoagulant, activated protein C (APC), aided by its cofactor protein S, cleaves the activated cofactors factor Va and factor VIIa, which are required in the intrinsic coagulation pathway to sustain thrombin formation (Esmon et al., Biochim. Biophys. Acta., 1477:349-360, 2000a), to yield the inactivated cofactors factor Vi and factor VIIIi.
The thrombin/thrombomodulin complex mediated activation of protein C is facilitated when protein C binds to the endothelial protein C receptor (EPCR), which localizes protein C to the endothelial cell membrane surface. When complexed with EPCR, APC's anticoagulant activity is inhibited; APC expresses its anticoagulant activity when it dissociates from EPCR, especially when bound to negatively charged phospholipids on activated platelet or endothelial cell membranes.
Components of the protein C pathway contribute not only to anticoagulant activity, but also to anti-inflammatory functions (Griffin et al., Sem. Hematology, 39:197-205, 2002). The anti-inflammatory effects of thrombomodulin, recently attributed to its lectin-like domain, can protect mice against neutrophil-mediated tissue damage (Conway et al., J. Exp. Med. 196:565-577, 2002). The murine centrosomal protein CCD41 or centrocyclin, involved in cell-cycle regulation is identical to murine EPCR lacking the first N-terminal 31 amino acids (Rothbarth et al., FEBS Lett., 458:77-80, 1999; Fukodome and Esmon, J. Biol. Chem., 270:5571-5577,1995). EPCR is structurally homologous to the MHC class 1/CD1 family of proteins, most of which are involved in inflammatory processes. This homology suggests that the function of EPCR may not be limited to its ability to localize APC or protein C on the endothelial membrane (Oganesyan et al., J. Biol. Chem., 277:24851-24854, 2002). APC provides EPCR-dependent protection against the lethal effects of E.coli infusion in baboons (Taylor et al., Blood, 95:1680-1686, 2000) and can downregulate proinflammatory cytokine production and favorably alter tissue factor expression or blood pressure in various models (Shu et al., FEBS Lett. 477:208-212, 2000; Isobe et al., Circulation, 104:1171-1175, 2001; Esmon, Ann. Med., 34:598-605, 2002).
Inflammation is the body's reaction to injury and infection. Three major events are involved in inflammation: (1) increased blood supply to the injured or infected area; (2) increased capillary permeability enabled by retraction of endothelial cells; and (3) migration of leukocytes out of the capillaries and into the surrounding tissue (hereinafter referred to as cellular infiltration) (Roitt et al., Immunology, Grower Medical Publishing, New York, 1989).
Many serious clinical conditions involve underlying inflammatory processes in humans. For example, multiple sclerosis (MS) is an inflammatory disease of the central nervous system. In MS, circulating leukocytes infiltrate inflamed brain endothelium and damage myelin, with resultant impaired nerve conduction and paralysis (Yednock et al., Nature 366:63-66 (1992)). Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of tissue damage caused by self antigen directed antibodies. Auto-antibodies bound to antigens in various organs lead to complement-mediated and inflammatory cell mediated tissue damage (Theofilopoulos, A.N., Encyclopedia of Immunology, pp. 1414-1417 (1992)).
APC has not only anticoagulant and anti-inflammatory activities but also anti-apoptotic activity. EPCR has been found to be a required cofactor for the anti-apoptotic activity of APC in certain cells, as APC activation of protease activated receptor-1 (PAR-1) is EPCR-dependent (Riewald et al., Science, 2296:1880-1882, 2002; Cheng et al., Nat. Med., 9:338-342, 2003; Mosnier and Griffin, Biochem. J., 373:65-70, 2003). APC also has been shown potentially to inhibit staurosporine-induced apoptosis in endothelial cells in vitro by modulating the expression of NFκB subunits (Joyce et al., J. Biol. Chem., 276:11199-11203, 2001). Staurosporine-induced apoptosis in human umbilical vein endothelial cells (HUVEC) and tumor necrosis factor-α-mediated injury of HUVEC, based on transcriptional profiling, suggest that APC's inhibition of NFκB signaling causes down regulation of adhesion molecules (Joyce et al., supra, 2001). APC's induction of anti-apoptotic genes (e.g., Bcl2-related protein A1 or Bcl2A1, inhibitor of apoptosis 1 or clAP1, endothelial nitric oxide synthase or eNOS) has been interpreted as a possible mechanism linked to APC's anti-apoptotic effects in a staurosporine model of apoptosis.
APC has a remarkable ability to reduce all-cause 28-day mortality by 19% in patients with severe sepsis (Bernard et al., New Engl. J. Med. 344:699-709, 2001a), whereas, potent anticoagulant agents such as antithrombin III and recombinant TFPI have failed in similar phase III clinical trials (Warren et al., JAMA, 286:1869-1878, 2001; Abraham et al., Crit. Care Med., 29:2081-2089). The explanation for this difference may lie in the recently described anti-apoptotic activity of APC, as well as its anti-inflammatory activity. The clinical success of APC in treating sepsis may be related to its direct cellular effects that mediate its anti-apoptotic or anti-inflammatory activity.
In spite of the numerous in vivo studies documenting the beneficial effects of APC, there is limited information about the molecular mechanisms responsible for APC's direct anti-inflammatory and anti-apoptotic effects on cells. APC can directly modulate gene expression in human umbilical vein endothelial cells (HUVEC) with notable effects on anti-inflammatory and cell survival genes (Joyce et al., supra, 2001; Riewald et al., supra, 2002). Riewald et al. have shown this direct effect of APC on certain cells requires PAR-1 and EPCR (Riewald et al., supra, 2002), although they provided no data that related APC functional activity with PAR-1-signaling.
Recombinant activated protein C (rAPC), similar to Xigris (Eli Lilly & Co.), is approved for treating severe sepsis and it may eventually have other beneficial applications. However, clinical studies have shown APC treatment to be associated with increased risk of serious bleeding. This increased risk of bleeding presents a major limitation of APC therapy. If APC's effects in sepsis can be attributed to its anti-inflammatory and cell survival activities, a compound that retains the beneficial anti-apoptotic or cytoprotective activity but has a less anticoagulant activity is desirable.